Modulation of TGF-β by recombinant TGF-β type II receptor polypeptides

ABSTRACT

The mammalian TGF-β type II receptor has been cloned. Recombinant TGF-β-binding receptor polypeptides are useful to alter the binding of TGF-β to its receptors and to modulate the effects of TGF-β in vivo.

FUNDING

Work described herein was funded by National Cancer Institute Grant No.R35-CA39826; National Heart, Lung and Blood Institute Centers ofExcellence Grant HL-41484; the Damon Runyon-Walter Winchell CancerResearch Fund; National Institutes of Health predoctoral training grantnumber T 32 BM07287-16; and the American Cancer Society. The UnitedStates government has certain rights in the invention.

This application is a division of application Ser. No. 08/311,703 filedSep. 23, 1994 which is a File Wrapper Continuation of 07/786,063, filedOct. 31, 1991, now abandoned.

BACKGROUND

Transforming growth factor-beta (TGF-β) is a member of a family ofstructurally related cytokines that elicit a variety of responses,including growth, differentiation, and morphogenesis, in many differentcell types. (Roberts, A. B. and H. B. Sporn, In: Peptide Growth Factorsand Their Receptors, Springer-Verlag, Heidelberg, pp. 421-472 (1990);Massague, J., Annu. Rev. Cell. Biol. 6: 597-641 (1990)) In vertebratesat least five different forms of TGF-β, termed TGF-β1 to TGF-β5, havebeen identified; they all share a high degree (60%-80%) of amino-acidsequence identity. While TGF-β1 was initially characterized by itsability to induce anchorage-independent growth of normal rat kidneycells, its effects on most cell types are anti-mitogenic. (Altschul, S.F. et al., J. Mol. Biol. 215: 403-410 (1990); Andres, J. L. et al., J.Cell. Biol. 109: 3137-3145 (1989)) It is strongly growth-inhibitory formany types of cells, including both normal and transformed epithelial,endothelial, fibroblast, neuronal, lymphoid, and hematopoietic cells. Inaddition, TGF-β plays a central role in regulating the formation ofextracellular matrix and cell-matrix adhesion processes.

In spite of its widespread effects on cell phenotype and physiology,little is known about the biochemical mechanisms that enable TGF-βfamily members to elicit these varied responses. Three distincthigh-affinity cell-surface TGF-β-binding proteins, termed type I, II andIII, have been identified by incubating cells with radiolabelled TGF-β1,cross-linking bound TGF-β1 to cell surface molecules, and analyzing thelabelled complexes by polyacrylamide gel electrophoresis. (Massague, J.and B. Like, J. Biol. Chem. 260: 2636-2645 (1985); Cheifetz, S. et al.J. Biol. Chem. 261: 9972-9978 (1986).) The binding constants are about5-50 pM for the type I and II receptor and 30-300 pM for the type IIIreceptor. (Boyd, F. T. and J. Massague, J. Biol. Chem. 264: 2272-2278(1989))

The type I and II receptors, of estimated 53 and 70-100 kilodaltons massrespectively, are N-glycosylated transmembrane proteins that are similarin many respects. Each of these receptors has a distinct affinity foreach member of the TGF-β family of ligands. (Boyd, F. T. and J.Massague, J. Biol. Chem. 264: 2272-2278 (1989)) In contrast, the typeIII receptor shows comparable affinities for all TGF-β isotypes; thetype III receptor is the most abundant cell-surface receptor for TGF-βin many cell lines (upwards of 200,000 per cell), and is an integralmembrane proteoglycan. It is heavily modified by glycosaminoglycan (GAG)groups, and migrates heterogeneously upon gel electrophoresis asproteins of 280 to 330 kilodaltons. When deglycosylated withheparitinase and chondrontinase, the protein core migrates as a 100-110kilodalton protein. The TGF-β binding site resides in this protein core,as non glycosylated forms of this receptor that are produced in cellmutants defective in GAG synthesis are capable of ligand binding withaffinities comparable to those of the natural receptor. (Cheifetz, S.and J. Massague, J. Biol. Chem., 264: 12025-12028 (1989) A variant formof type III receptor is secreted by some types of cells as a solublemolecule that apparently lacks a membrane anchor. This soluble speciesis found in low amounts in serum and in extracellular matrix.

The type III receptor, also called betaglycan, has a biological functiondistinct from that of the type I and II receptors. Some mutant mink lungepithelial cell (Mv1Lu) selected for loss of TGF-β responsiveness nolonger express type I receptors; others, similarly selected, loseexpression of both the type I and II receptors. However, all thesevariants continue to express the type III receptor. (Boyd, F. T. and J.Hassague, J. Biol. Chem. 264: 2272-2278 (1989); Laiho, M. et al., J.Biol. Chem. 265: 18518-18524 (1990)) This has led to the proposal thattypes I and II receptors are signal-transducing molecules while the typeIII receptor, may subserve some other function, such as in concentratingligand before presentation to the bona fide signal-transducingreceptors. The secreted form of type III receptor, on the other hand,may act as a reservoir or clearance system for bioactive TGF-β.

Additional information about-each of these TGF-β receptor types wouldenhance our understanding of their roles and make it possible, ifdesired, to alter their functions.

SUMMARY OF THE INVENTION

The present invention relates to isolation, sequencing andcharacterization of DNA encoding the TGF-β type III receptor ofmammalian origin and DNA encoding the TGF-β type II receptor ofmammalian origin. It also relates to the encoded TGF-β type III and typeII receptors, as well as to the soluble form of each; uses of thereceptor-encoding genes and of the receptors themselves; antibodiesspecific for TGF-β type III receptor and antibodies specific for TGF-βtype II receptor. In particular, it relates to DNA encoding the TGF-βtype III receptor of rat and human origin, DNA encoding the TGF-β typeII receptor of human origin and homologous of each.

The TGF-β receptor-encoding DNA of the present invention can be used-toidentify equivalent TGF-β receptor type III and type II genes from othersources, using, for example, known hybridization-based methods or thepolymerase chain reaction. The type III receptor gene, the type IIreceptor gene or their respective encoded products can be used to alterthe effects of TGF-β (e.g., by altering receptivity of cells to TGF-β orinterfering with binding of TGF-β to its receptor), such as its effectson cell proliferation or growth, cell adhesion and cell phenotype. Forexample, the TGF-β receptor type III gene, the TGF-β receptor type IIgene, or a truncated gene which encodes less than the entire receptor(e.g., soluble TGF-β type III receptor, soluble TGF-β type II receptoror the TGF-β type III or type II binding site) can be administered to anindividual in whom TGF-β effects are to be altered. Alternatively, theTGF-β type III receptor, the TGF-β type II receptor, a soluble formthereof (i.e., a form lacking the membrane anchor) or an active bindingsite of the TGF-β type III or the type II receptor can be administeredto an individual to alter the effects of TGF-β.

Because of the many roles TGF-β has in the body, availability of theTGF-β receptors described herein makes it possible to further assessTGF-β function and to alter (enhance or diminish) its effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C (SEQ ID NOS: 5 and 6) is the DNA sequence and the translatedamino acid sequence of type III TGF-β1 receptor cDNA clone R3-OFF (fullinsert size 6 kb), in which the open reading frame with flankingsequences of the clone are shown. The transmembrane domain is indicatedby a single underline. Peptide sequences from purified type IIIreceptor, mentioned in text, that correspond to the derived sequence,are in italics and underlined. Potential N-linked glycosylation sitesare indicated by #, and extracellular cysteines by &. A consensusprotein kinase C phosphorylation site is indicated by $. The lastnon-vectorencoded amino acid of Clone R3-OF (2.9 kb) is indicated by @.Consensus proteoglycan attachment site is indicated by +++. Otherpotential glycosaminoglycan attachment sites are indicated by +. Theupstream in-frame stop codon (-42 to -44) is indicated by a wavy line.Signal peptide cleavage site predicted by vonHeijne's algorithm (vonHeijne, G., Nucl. Acid. Res. 14: 4683-4690 (1986) is indicated by anarrow.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention is based on the isolation and sequencing of DNA ofvertebrate, particularly mammalian, origin which encodes TGF-β type IIIreceptor and DNA of mammalian origin which encodes TGF-β type IIreceptor, expression of the encoded products and characterization of theexpressed products. As described, a full-length cDNA which encodes TGF-βreceptor type III has been isolated from a cDNA library constructed froma rat vascular smooth muscle cell line and a full-length cDNA whichencodes TGF-β type II receptor has been isolated from a human cDNAlibrary. The human homologue of the type III gene has also been cloned.A deposit of human TGF-β type III cDNA in the plasmid pBSK has been madeunder the terms of the Budapest Treaty at the American Type CultureCollection (Oct. 21, 1991) under Accession Number 75127. Allrestrictions upon the availability of the deposited material will beirrevocably removed upon granting of a U.S. patent based on the subjectapplication.

Isolation and Characterization of TGF-β Type III Receptor

As described herein, two separate strategies were pursued for theisolation of the TGF-β type III receptor cDNA. In one approach,monoclonal antibodies were generated against the type III receptorprotein and used to purify the receptor, which was then subjected tomicrosequencing. (See Example 1) Microsequencing of several peptidesresulting from partial proteolysis of the purified receptor producedfour oligopeptide sequences, which were used to construct degenerateoligonucleotides. The degenerate oligonucleotides were used either asprimers in a cloning strategy using the polymerase chain reaction (PCR)or as probes in screening cDNA libraries. Although this strategy did notprove to be productive, the oligopeptide sequences were useful inverifying the identity of the receptor clones isolated by the secondstrategy.

In the second approach to isolating TGF-β receptor-encoding clones, anexpression cloning strategy was used in COS cells; direct visualizationof receptor positive cells was used to isolate receptor cDNAs. (SeeExample 2) In this approach, a cDNA library was constructed from A-10cells, a rat vascular smooth muscle cell line which expresses all threeTGF-β receptors (type I, II and III). COS cells transfected with cDNAcomponents of this library in a vector carrying the cytomegalovirus(CMV) transcriptional promoter and the SV40 origin of replication werescreened to identify cells expressing substantially higher than normallevels of TGF-β receptor. One transfectant expressing such high levelsof a TGF-β binding protein was identified and the original pool ofexpression constructs from which it was derived was split into subpools,which were subjected to a second round of screening. Two further roundsof sib-selection resulted in isolation of one cDNA clone (R3-OF) with a2.9 kb insert which induced high levels of TGF-β-binding proteins inapproximately 10% of cells into which it was introduced. The specificityof the TGF-β binding was validated by showing that addition of a200-fold excess unlabeled competitor TGF-β1 strongly reduced binding of125 I-TGF-β to transfected cells.

The R3-OF cDNA encoded an open reading frame of 817 amino acid residues,but did not contain a stop codon. R3-OF was used as a probe to isolate afull-length cDNA from a rat 208F library. The resulting clone, R3-OFF,is 6 kb in length and encodes a protein of 853 amino acids, which iscolinear with clone R3-OF. The nucleotide sequence of R3-OFF is shown inFIGS. 1A-1C, along with the translated amino acid sequence.

Characterization of the receptor encoded by R3-OFF was carried out, asdescribed in Example 3. Results showed three distinct TGF-β bindingprotein species of TGF-β on the surface of mock-transfected COS cells,which is in accord with results reported by others. (Massague, J. etal., Ann. NY Acad. Sci. 593: 59-72 (1990)). These included the two lowermolecular weight type I and II receptors (65 and 85 kD) and the highermolecular weight type III proteoglycan, which migrates as a diffuse bandof 280-330 kd. Enzymatic removal of the proteoglycan yielded a coreprotein of approximately 100 kd. Binding to all three receptor types isspecific in that it was competed by 200-fold excess of unlabeled TGF-β1.

Transfecting the isolated cDNA caused a two-fold increase in expressionof the type III receptor. When a cell lysate derived from COS cellstransfected with clone R3-OFF was treated with deglycosylating enzymes,the heterogeneous 280-330 kd band was converted to a protein core whichco-migrates with the type III protein core seen in parental A10 cells.Importantly, the recombinant protein core migrated differently from theendogenous COS cell type III protein core.

These observations were confirmed and extended using stably transfectedcells expressing the type III cDNA. L6 rat skeleton muscle myoblasts donot express any detectable type III mRNA and no endogeneous surface typeIII receptor (Massague et al., 1986; Segarini et al., 1989). These cellswere transfected with the isolated cDNA in the vector pcDNA-neo. Cellclones stably expressing this clone in both the forward and reverseorientations with respect to the CMV promoter were isolated and analyzedby ligand binding assay.

Introduction of either the full-length clone R3-OFF or the partial cloneR3-OF in the forward orientation resulted in expression of type IIIreceptor. L6 cells transfected with the cDNA clones in the reverseorientation did not express this protein. Importantly, the apparent sizeof the protein core of the type III receptor in cells transformed withthe R3-OF clone is smaller than that from R3-OFF transformed cells,consistent with the difference in the sizes of the protein corespredicted from their nucleic acid sequences.

Surprisingly, binding of radio-labeled ligand to the type II receptorwas increased by 2.5 fold in cells expressing the type III cDNA. Bindingto the type I receptor was unchanged. This apparently specificup-regulation of ligand-binding to the type II receptor was evident inall of the 15 stably transfected L6 cell lines analyzed to date.Furthermore, this effect seems to be mediated equally well by thefull-length clone or a truncated clone (R3-OF) that lacks thecytoplasmic domain of TGF-β type III receptor was expressed.

Expression of type III receptor mRNA was assessed by Northern blotanalysis and RNA blot analysis. Northern gel analysis showed that thetype III receptor mRNA is expressed as a single 6 kb message in severalrat tissues. RNA dot blot analysis of several different tissue culturecell lines was also carried out. Cells of mouse origin (MEL and YH16)appear to express a smaller (˜5.5 kb) message for-the type III mRNA thanthose of pig, rat and human origin. In all of these cells, expression orabsence of the type III mRNA is consistent with the expression orabsence of detectable cell surface type III receptors, with the notableexception of the retinoblastoma cell lines (Y79, Weri-1, Weri-24, andWeri-27). These cells lack detectable surface expression of type IIIreceptor, which confirms an earlier report. (Kimchi, A. et al., Science240: 196-198 (1988)). It is striking that the type III receptor MRNA isexpressed in these cells at a level comparable to that of other cellsthat do indeed express type III receptor proteins at readily detectablelevels. It appears that TGF-β receptor III expression, which issubstantial in normal retinoblasts (AD12), has been down-regulated inthese retinoblastoma tumor cells, perhaps through post-transcriptionalmechanisms.

The nucleotide sequence full reading frame along with flanking sequencesof the full-length cDNA clone R3-OFF was determined and is presented inFIGS. 1A-1C. The reading frame encodes a protein of 853 amino acidresidues, which is compatible with the 100 kD size observed for thefully deglycosylated TGF-β1 type III receptor. The identity of thereceptor as TGF-β type III was verified by searching for segments of theputative transcription product which included the peptide sequencesdetermined by microsequencing of the isolated type III receptor. (SeeExample 1) As indicated in FIGS. 1A-1C, two segments of derived protein(underlined and italicized, residues 378-388 and 427-434) preciselymatch with the amino acid sequences of two peptides (I and III)determined from direct biochemical analysis of the purified type IIIreceptor.

Further analysis showed that TGF-β type III binding protein has anunusual structure for a cytokine receptor. Hydropathy analysis indicatesthat the protein includes a N-terminal signal sequence, followed by along, hydrophilic N-terminal region. A 27 residue region of stronghydrophobicity (underlined in FIGS. 1A-1C, residues 786-812) toward theC-terminus represents the single putative transmembrane domain. Thissuggests that nearly all of the receptor which is an N-terminalextracellular domain is anchored to the plasma membrane near itsC-terminus. A relatively small C-terminal tail of 41 residues representsthe cytoplasmic domain.

Analysis of related sequences provides few clues to function of TGF-βtype III protein. Only one other gene described to date, a glycoproteinexpressed in high quantities by endothelial cells and termed endoglin,contains a related amino acid sequence. The most homologous regionsbetween the sequences of the type III receptor and endoglin (74%) fallsprimarily in the putative transmembrane and cytoplasmic domains. Similarto the general structure of type III receptor, endoglin is aglycoprotein which contains a large hydrophilic N-terminal domain whichis presumably extracellular, followed by a putative transmembrane domainand a short cytoplasmic tail of 47 amino acid residues. The biologicalrole of endoglin is still unclear at present, although it has beensuggested that it may involved in cell-cell recognition throughinteractions of an "RGD" sequence on its ectodomain with other adhesionmolecules. Unlike the TGF-β type III receptor, endoglin does not carryGAG groups.

Isolation of TGF-β Type II Receptor

The cDNA encoding the type II TGF-β receptor was also isolated, usingexpression cloning in COS cells. A full-length cDNA (designated clone3FF) was isolated by high stringency hybridization from a human HepG2cell cDNA library. Analysis showed that the corresponding message is a 5kb message which is expressed in different cell lines and tissues.Sequence analysis indicated that the cDNA has an open reading frameencoding a core 567 amino acid residue protein. The nucleotide sequenceof the full-length type II TGF-β receptor cDNA clone 3FF is shown in SEQID NO: 7; the amino acid sequence is represented in SEQ ID NO: 8.

The 567 amino acid residue protein has a single putative transmembranedomain, several consensus glycosylation sites, and a putativeintracellular serine/threonine kinase domain. The predicted size of theencoded protein core is ˜60 kd, which is too large for a type I TGF-βreceptor. Instead, crosslinking experiments using iodinated TGF-β andCOS cells transiently transfected with clone 3FF shows over-expressionof a protein approximately 70-80 kd which corresponds to the size oftype II TGF-β receptors. Thus, clone 3FF encodes a protein thatspecifically binds TGF-β and has an expressed protein size of 70-80 kd,both characteristic of the type II TGF-β receptor.

Plasmid 3FF was deposited in accordance with the provisions of theBudapest Treaty at the American Type Cilture Collection (ATCC), 12301Parklawn Drive, Rockville, Md. 20852, U.S.A. on Nov. 12, 1997, andassigned Accession Number ATCC 209455.

Uses of the Cloned TGF-β Receptors and Related Products

For the first time, as a result of the work described herein, DNAsencoding two of the three high affinity cell-surface TGF-β receptorshave been isolated, their sequences and expression patterns determinedand the encoded proteins characterized. Expression of the TGF-β type IIIreceptor in cells which do not normally express the receptor, followedby ligand binding assay, verified that the cloned type IIIreceptor-encoding DNA (i.e., either the full-length clone R3-OFF or thepartial clone R3-OF) encoded the receptor. In addition, the workdescribed herein resulted in the surprising finding that binding ofTGF-β to type II receptors in cells expressing the type III DNA wasincreased by 2.5 fold.

Additional insight into the role of the TGF-β type III receptor and itsinteraction with TGF-β type II receptor is a result of the workdescribed. For example, the role of TGF-β type III receptor is unclear,but it has been proposed that it serves a most unusual function ofattracting and concentrating TGF-βs for eventual transfer to closelysituated signal-transducing receptors. While most cytokines bind to asingle cell surface receptor, members of the TGF-β family bind withgreater or lesser affinity to three distinct cell surface proteins. Thishas raised the question of why these three receptors are displayed bymost cell types and whether they subserve distinct functions. Evidenceobtained to date suggests that the type III receptor may performfunctions quite different from those of types I and II. Thus, type IIIis substantially modified by GAGs while types I and II appear to carryprimarily the N-linked (and perhaps O-linked) sidechains that arecharacteristic of most growth factor receptors. In addition, variantcells that have been selected for their ability to resist TGF-β-inducedgrowth inhibition show the absence of Type I or Type II receptors whilecontinuing to display Type III receptors. Together, these data havecaused some to propose that the Type I and II receptors represent bonafide signal-transducing receptors while the type III receptor, describedhere, plays another distinct role in the cell.

It remains possible that the type III receptor serves a most unusualfunction of attracting and concentrating TGF-βs on the cell surface foreventual transfer to closely situated signal-transducing receptors. Sucha function would be unprecedented for a proteinaceous receptor, althoughheparin sulfate has been shown to activate basic FGF by binding to thisgrowth factor prior to FGF association with its receptor (Yayon, A. etal., Cell 64: 841-848 (1991)) Parenthetically, since the type IIIreceptor also contains large quantities of heparan sulfate side-chains,it may also bind and present basic FGF to its receptor.

Evidence that is consistent with the role for the type III receptorcomes from the work with L6 rat myoblast cells which is describedherein. As described above, in L6 cells overexpressing type IIIreceptor, the binding of radiolabelled TGF-β to the type II receptor isincreased several fold when compared with that seen with parental cells.Further assessment of TGF-β type III function and interaction with typeII and type I receptors will be needed to answer these questions and canbe carried out using the materials and methods described here.

TGF-β receptors, both type III and type II, can be identified in otherspecies, using all or a portion of the DNA encoding the receptor to beidentified as a probe and methods described herein. For example, all ora portion of the DNA sequence encoding TGF-β type III receptor (shown inFIGS. 1A-1C) or all or a portion of the DNA sequence encoding TGF-β typeII receptor (shown in SEQ ID NO: 7) can be used to identify equivalentsequences in other animals. Stringency conditions used can be varied, asneeded, to identify equivalent sequences in other species. Once aputative TGF-β receptor type III or type II-encoding sequence has beenidentified, whether it encodes the respective receptor type can bedetermined using known methods, such as described herein forverification that the cDNA insert of full-length clone R3-OFF and thecDNA insert of partial clone R3-OF encode the type III receptor. Forexample, DNA isolated in this manner can be expressed in an appropriatehost cell which does not express the receptor mRNA or the surfacereceptor (e.g., L6 rat skeleton muscle myoblasts) and analyzed by ligandbinding (TGF-β binding) assay, as described herein.

Also as a result of the work described herein, antibodies (polyclonal ormonoclonal) specific for the cloned TGF-β type III or the clones TGF-βtype II receptor can be produced, using known methods. Such antibodiesand host cells (e.g., hybridoma cells) producing the antibodies are alsothe subject of the present invention. Antibodies specific for the clonedTGF-β receptor can be used to identify host cells expressing isolatedDNA thought to encode a TGF-β receptor. In addition, antibodies can beused to block or inhibit TGF-β activity. For example, antibodiesspecific for the cloned TGF-β type III receptor can be used to blockbinding of TGF-β to the receptor. They can be administered to anindividual for whom reduction of TGF-β binding is desirable, such as insome fibrotic disease (e.g., of skin, kidney and lung).

DNA and RNA encoding TGF-β type III receptor and DNA and RNA encodingTGF-β type II receptor are now available. As used herein, the term DNAor RNA encoding the respective TGF-β receptor includes anyoligodeoxynucleotide or oligodeoxyribonucleotide sequence which, uponexpression, results in production of a TGF-β receptor having thefunctional characteristics of the TGF-β receptor. That is, the presentinvention includes DNA and RNA which, upon expression in an appropriatehost cell, produces a TGF-β type III receptor which has an affinity forTGF-β similar to that of the TGF-β type III receptor on naturallyoccurring cell surfaces (e.g., it shows comparable affinities for allTGF-β isotypes). Similarly, the present invention includes DNA and RNAwhich, upon expression in an appropriate host cell, produces a TGF-βtype II receptor which has an affinity for TGF-β similar to that ofTGF-β type II receptor on naturally occurring cell surfaces (e.g., ithas a distinctive affinity for each member of the TGF-β family ofligands similar to that of the naturally occurring TGF-β type IIreceptor). The DNA or RNA can be produced in an appropriate host cell orcan be produced synthetically (e.g., by an amplification technique suchas PCR) or chemically.

The present invention also includes the isolated TGF-β type III receptorencoded by the nucleotide sequence of full-length R3-OFF, the isolatedTGF-β type III receptor encoded by the nucleotide sequence of partialclone R3-OF, the isolated TGF-β type II receptor encoded by thenucleotide sequence of full-length clone 3FF and TGF-β type III and typeII receptors which bind TGF-β isotypes with substantially the sameaffinity. The isolated TGF-β type III and type II receptors can beproduced by recombinant techniques, as described herein, or can beisolated from sources in which they occur naturally or synthesizedchemically. As used herein, the terms cloned TGF-β type III and clonedTGF-β type II receptors include the respective receptors identified asdescribed herein, and TGF-β type III and type II receptors (e.g., fromother species) which exhibit substantially the same affinity for theTGF-β isotypes as the respective receptors.

As described previously, cells in which the cloned TGF-β type IIIreceptor is expressed bind TGF-β in essentially the same manner as docells on which the type III receptor occurs naturally. Further analysisof ligand interactions with the cloned TGF-β type III receptor, basedupon site-directed mutagenesis of both TGF-β and the receptor, can becarried out to identify residues important for binding. For example, DNAhaving the sequence of FIGS. 1A-1C can be altered by adding, deleting orsubstituting at least one nucleotide, in order to produce a modified DNAsequence which encodes a modified cloned TGF-β type III receptor. Thefunctional characteristics of the modified receptor (e.g., itsTGF-β-binding ability and association of the binding with effectsnormally resulting from binding) can be assessed, using the methodsdescribed herein. Modification of the cloned TGF-β type III receptor canbe carried out to produce, for example, a form of the TGF-β type IIIreceptor, referred to herein as soluble TGF-β receptor, which is notmembrane bound and retains the ability to bind the TGF-β isotypes withan affinity substantially the same as the naturally-occurring receptor.Such a TGF-β type III receptor could be produced, using known geneticengineering or synthetic techniques; it could include none of thetransmembrane region present in the naturally-occurring TGF-β type IIIreceptor or only a small portion of that region (i.e., small enough notto interfere with its soluble nature). For example, it can include aminoacids 1 through 785 of the TGF-β type III sequence of FIGS. 1A-1C or aportion of that sequence sufficient to retain TGF-β binding ability(e.g., amino acids 24-785, which does not include the signal peptidecleavage site present in the first 23 amino acids). A soluble TGF-β typeII receptor (e.g., one which does not include the transmembrane andcytoplasmic domains) can also be produced. For example, it can includeamino acids 1 through 166, inclusive, of SEQ ID NO: 8 or a sufficientportion thereof to retain TGF-β binding ability substantially the sameas that of TGF-β type II receptor.

The TGF-β type III receptor and/or type II receptor can be used fortherapeutic purposes. As described above, the TGF-β family of proteinsmediate a wide variety of cellular activities, including regulation ofcell growth, regulation of cell differentiation and control of cellmetabolism. TGF-β may be essential to cell function and most cellssynthesize TGF-β and have TGF-β cell surface receptors. Depending oncell type and environment, the effects of TGF-β vary: proliferation canbe stimulated or inhibited, differentiation can be induced orinterrupted and cell functions can be stimulated or suppressed. TGF-β ispresent from embryonic stages through adult life and, thus, can affectthese key processes throughout life. The similarities of a particularTGF-β (e.g., TGF-β1) across species and from cell to cell areconsiderable. For example, the amino acid sequence of a particular TGF-βand the nucleotide sequence of the gene which encodes it regardless ofsource, are essentially identical across species. This further suggeststhat TGF-β has a critical role in essential processes.

Specifically, TGF-β has been shown to have anti-inflammatory and immunesuppression capabilities, to play an important role in bone formation(by increasing osteoblast activity), inhibit cancer cell proliferationin culture, and control proliferation of glandular cells of theprostate. As a result, it has potential therapeutic applications inaltering certain immune system responses (and possibly in modifyingimmune-mediated diseases); in treating systemic bone disease (e.g.,osteoporosis) and conditions in which bone growth is to be enhanced(e.g., repair of broken bones) and in controlling growth and metastasisof cancer cells. In addition, TGF-β appears to play a role indetermining whether some cell types undergo or do not undergo mitosis.In this respect, TGF-β may play an important role in tissue repair. Somediseases or conditions appear to involve low production or chronicoverproduction of TGF-β. (For example, results of animal studies suggestthat there is a correlation between the over production of TGF-β anddiseases characterized by fibrosis in the lung, kidney, liver or inviral mediated immune expression.)

Clearly, TGF-β has key roles in body processes and numerous relatedpotential clinical or therapeutic applications in wound healing, cancer,immune therapy and bone therapy. Availability of TGF-β receptor genes,the encoded products and methods of using them in vitro and in vivoprovides an additional ability to control or regulate TGF-β activity andeffect in the body. For example, the TGF-β type II or type III receptorencoded by the type II or the type III receptor genes of the subjectinvention can be used, as appropriate, to alter the effects of TGF-β(e.g., to enhance the effect of TGF-β in the body or to inhibit orreduce (totally or partially) its effects). It is also possible toadminister to an individual in whom TGF-β bound to TGF-β type IIIreceptor, such as soluble TGF-β type III receptor. The present inventionprovides both a TGF-β agonist and a TGF-β antagonist. For thesepurposes, DNA gene encoding the entire TGF-β type II or type IIIreceptor, the encoded type II or type III receptor or a soluble form ofeither receptor can be used. Alternatively, antibodies or other ligandsdesigned based upon these sequences or specific for them can be used forthis purpose.

Knowledge of the amino acid sequences of TGF-β type III and type IIreceptors makes it possible to better understand their structure and todesign compounds which interfere with binding of the receptor withTGF-β. It makes possible identification of existing compounds and designof new compounds which are type III and/or type II receptor antagonists.

Cells expressing the type III and/or type II receptors of the presentinvention can be used to screen compounds for their ability to interferewith (block totally or partially) TGF binding to the receptors. Forexample, cells which do not express TGF-β type III receptor (e.g., L6rat skeleton muscle myoblasts) but have been modified to do so byincorporation of the type III cDNA in an appropriate vector can be usedfor this purpose. A compound to be assessed is added, for example, totissue culture dishes containing type III expressing cells, along withlabeled TGF-β. As a control, the same concentration of labeled TGF-β isadded to tissue culture dishes containing the same type of cells. Aftersufficient time for binding of TGF-β to the receptor to occur, bindingof labeled TGF-β to the cells is assessed, using known methods (e.g., bymeans of a gamma counter) and the extent to which it occurred in thepresence and in the absence of the compound to be assessed isdetermined. Comparison of the two values show whether the test compoundblocked TGF-β binding to the receptor (i.e., less binding in thepresence of the compound than in its absence is evidence that the testcompound has blocked binding of TGF-β to the TGF-β type III receptor).

Alternatively, a cell line expressing the TGF-β receptor or cellsexpressing microinjected TGF-β receptor RNA, can be used to assesscompounds for their ability to block TGF-β binding to the receptor. Inthis embodiment, a compound to be assessed is added to tissue culturedishes containing the cell line cells expressing microinjected TGF-βreceptor RNA, along with TGF-β. As a control, TGF-β alone is added tothe same type of cells expressing microinjected endothelin receptor RNA.After sufficient time for binding of TGF-β to the receptor to occur, theextent to which binding occurred is measured, both in the presence andin the absence of the compound to be assessed. Comparison of the twovalues shows whether the compound blocked TGF-β binding to the receptor.The TGF-β type III and type II receptors can also be used to identifyTGF-β-like substances, to purify TGF-β and to identify TGF-β regionswhich are responsible for binding to the respective receptors. Forexample, the type III receptor can be used in an affinity-based methodto identify substances which bind the receptor in a manner similar toTGF-β.

The invention will now be illustrated by the following examples, whichare not intended to be limiting in any way.

EXAMPLES

Materials and methods used in Examples 1-5 are described below.

Materials

The following is a description of materials used in the work describedherein.

Recombinant human TGF-β1 was provided by Rik Derynck of Genentech.COS-M6 cells were provided by Brian Seed of the Massachusetts GeneralHospital and Alejandro Aruffo of Bristol-Myers-Squibb. Heparitinase wasprovided by Tetsuhito Kojima and Robert Rosenberg of MIT. LLC-PK_(l)cells were a gift of Dennis Ausiello of the Massachusetts GeneralHospital. YH-16 cell were a gift of Edward Yeh of the MassachusettsGeneral Hospital. 3-4 cells were a gift of Eugene Kaji of the WhiteheadInstitute for Biomedical Research. All other cell lines were purchasedfrom ATCC and grown as specified by the supplier, except as noted.

Expression Cloning

Construction of CDNA Library and Generation of Plasmid Pools

10 μg polyadenylated mRNA was prepared from A10 cells by theproteinase-K/SDS method (Gonda et al., Molec. Cell. Biol. 2: 617-624(1982)). Double stranded cDNA was synthesized and linkered tononpalindromic BstXl adaptors as described by Seed, B. and A. Aruffo,Proc. Natl. Acad. Sci. USA 84: 3365-3369 (1987). Acaptored CDNA wassize-fractionated on a 5 to 20% potassium acetate gradient, and insertsgreater than 1 kb were ligated to the plasmid vector pcDNA-1, andelectroporated in the E. coli MC1061/P3, yielding a primary library witha titer of >10⁷ recombinants. A portion of the cDNA was plated as poolsof ˜1×10⁴ recombinant bacteria colonies grown on 15 cm petri dishes withLuria-broth agar containing 7.5 mg/ml tetracycline and 12.5 mg/mlampicillin. Cells were scraped off the plates in 10 mls of Luria-broth,and glycerol stocks of pooled bacteria were stored at -70° C. Theremaining bacteria was used to purify plasmid DNA using the alkalinelysis mini-prep method (Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2d Ed. (Cold Spring Harbor, N.Y., Cold Spring HarborLaboratory Press (1989)).

COS Cell Transfections and Binding Assay

Plasmid pools (each representing ˜1×10⁴ clones) were transfected intoCOS-M6 (subelone of COS-7 cells) by the DEAE-dextran/chloroquine methoddescribed by Seed, B. and A. Aruffo, Proc. Natl. Acad. Sci. USA 84:3365-3369 (1987). Forty-eight hours after transfection, cells wereincubated with 50 p¹²⁵ I-TGF-β1 (100 to 200 Ci/mmol) for 4 hours at 4°C.), autoradiographic analysis of transfected cells was performed usingNT-B2 photographic emulsion (Kodak) essentially as described (Gearing,D. P. et al., EMBO J. 8: 3667-3676 (1989)). After development of slides,cells were air-dried and mounted with mounting media and a glasscoverslip. Slides were analyzed under an Olympus 0M-T1 invertedphase-contrast microscope using a dissection trans-illuminator fordarkfield illumination.

Subdivision of Positive Pool

Of 86 pools screened, one pool (#13) was identified as positive and aglycerol stock of bacteria corresponding to this pool was titered and 25pools of 1000 clones were generated and miniprep plasmid from thesepools were transfected into COS cells as described above. Severalpositive pools of 1000 were identified, and one was replated as 25plates of 100 colonies. A replica was made of this positive-plate andharvested. Once a positive pool was identified, individual colonies werepicked from the corresponding master plate and grown overnight in 3 mlliquid culture. A 2-dimensional grid representing the 100 clones wasgenerated and a single clone, R3-OF, was isolated.

Cloning of R3-OFF

A 208F rat fibroblast library in lambda ZAP II (Stratagene) was screenedat high stringency with clone R3-OF insert, and several clones with ˜6kb inserts were isolated, one of which is referred to as R3-OFF.

DNA Sequencing and Squence Analysis

Double-stranded DNA was sequenced by the dideoxy chain terminationmethod using Sequenase reagents (United States Biochemicals). Comparisonof the sequence to the data bases was performed using BLAST (Altschcul,S. F. et al., J. Mol. Biol. 215: 403-410 (1990)).

Iodination of TGF-β

TGF-β1 was iodinated using the chloramine-T method as described(Cheifetz, S. and J. L. Andres, J. Biol. Chem. 263: 16984-16991 (1988)).

Chemical Cross-Linking

Transfected COS cells grown on 10 cm dishes or subconfluent L6 and A-10cells grown on 3.5 cm dishes were incubated with ¹²⁵ I-TGF-β1 in bindingbuffer (Frebs-Ringer buffered with 20 mM Hepes, pH 7.5, 5 mM mgSO₄, 0.5%BSA), washed 4 times with ice-cold binding buffer without BSA, andincubated for 15 minutes with binding buffer without BSA containing 60ng/ml disuccinimidyl suberate at 4° C. under constant rotation.Crosslinking was terminated by addition of 7% sucrose in binding buffer.Cells were scraped, collected and pelleted by centrifugation, thenresuspended in lysis buffer (10 mM Tris, pH 7.4, 1 mM EDTA, pH 8.0, 1%Triton-X 100, 10 μg/ml of pepstatin, 10 μg/ml leupeptin, 10 μg/mlantipain, 100 μg/m; benzamidine hydrochloride, 100 μg/ml soybean trypsininhibitor, 50 μg/ml aprotonin, and 1 mM phenylmethylsulfonyl fluoride).Solubilized material was analyzed by 7% SDS-PAGE and subjected toautoradiographic analysis by exposure to X-AR film (Kodak) at -70° C.

Enzymatic Digestion

Digestion of solubilized TGF-b receptors with chondroitinase andheparitinase was performed as described (Cheifetz, S. and J. L. Andres,J. Biol. Chem. 263: 16984-16991 (1988); Segarini, P. R. and S. M.Seyedin, J. Biol. Chem., 263: 8366-8370 (1988).

Generation of Stable Cell Lines

L6 myoblasts were split 1:10 into 10 cm dishes and transfected thefollowing day by the calcium phosphate method (Chen, C. and H. Okayama,Molec. Cell. Biol. 7: 2745-2752 (1987)) with clones R3-OF or R3-OFF inthe forward and reverse orientations in the vector pcDNA-neo(InVitrogen). Cells were subjected to selection in the presence of G418(Geneticin, GIBCO) for several weeks until individual colonies werevisible by the naked eye. These clones were isolated and amplified.

RNA Blot Analyses

Rat tissue polyadenylated MRNA was prepared by the lithium chloride/ureamethod (Auffrey, C. and F. Raugeon, Eur. J. Biochemistry 107: 303-313(1980), followed by oligo-dT cellulose chromatography (Aviv and Leder,1972). Polyadenylated mRNA from cell lines was prepared by theproteinase K/SDS method (Gonda, T. J. et al., Molec. Cell. Biol. 2:617-624 (1982)). Samples of mRNA were resolved by electrophoresis on 1%agarose-2.2M formaldehyde gels, blotted onto nylon membranes (Biotrns,ICN) and incubated with the 2.9 kb insert of clone Re-OF labeled with ³²p by random priming as probe (Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2d Ed., Cold Spring Harbor, N.Y., Cold Spring HarborLaboratory Press, (1989). Hybridizations were performed at 42° C. inhybridization buffer containing 50% formamide overnight, and blots werewashed at 55° C. in 0.2× SSC, 0.1% SDS, before exposure to X-AR film at-70° C.

Example 1 Production of Anti-Type III Receptor Protein Antibodies andMicrosequencing and Micro-sequencing of Peptides Resulting from PartialProteolysis of Purified Type III Receptor

Initially cellular proteoglycans were purified from human placenta andthen subjected to enzymatic deglycosylation with heparitinase andchondroitinase. Protein cores in the molecular weight range of 100-130kilodaltons were further purified by preparative gel electrophoresis;these should include the type III receptor. This partially purifiedmaterial was used as an immunogen in mice. After screening 850 hybridomalines developed from immunized mice, three lines were found to produceantibodies that specifically recognized and immunoprecipitated adeglycosylated polypeptide species of 100-120 kD. This species could beradiolabelled by incubation of whole cells with ¹²⁵ I-TGF-β followed bycovalent cross-linking. Its size is consistent with that of the proteincore previously reported for the type III receptor. (Massague, J., Annu.Rev. Cell. Biol. 6: 597-641 (1990))

Monoclonal antibody 94 was used to purify the type III receptor from ratliver by affinity-chromatography. The purified receptor was subjected topartial proteolysis and the resulting peptides were resolved by highpressure liquid chromatography. Several peptides were subjected tomicrosequencing and yielded the following oligopeptide sequences:

Peptide I: ILLDPDHPPAL (SEQ ID NO: 1)

Peptide II: QAPFPINFMIA (SEQ ID NO: 2)

Peptide III: QPIVPSVQ (SEQ ID NO: 3)

Peptide IV: FYVEQGYGR (SEQ ID NO: 4)

These peptide sequences were used to construct degenerateoligonucleotides that served either as primers in a cloning strategyusing the polymerase chain reaction (PCR) or as probes in screening cDNAlibraries. While this strategy was not productive, the oligopeptidesequences proved useful in verifying the receptor clones isolated by thesecond, alternative strategy (see Example 2).

Example 2 Expression Cloning of the Type III Receptor cDNA

An expression cloning strategy in COS cells, a procedure which takesadvantage of the considerable amplification of individual cDNAs intransfected COS cells was used as an alternative means to isolate TGF-βreceptor clones. Such amplification is mediated by SV40 large T antigenexpressed by the COS cells interacting with a SV40 origin of replicationin the CDNA vector. Gearing, D. et al., EMBO J. 8: 3667-3676 (1989);Lin, H. Y., et al., Proc. Natl. Acad. Sci. 88: 3185-3189 (1991a); Lin,H. Y. et al., Science, in press (1991); Mathews, L. S. and Vale, W. W.,Cell 65: 973-982 (1991).

The strategy involved the construction of a cDNA library from A-10cells, a rat vascular smooth muscle cell line that expresses all threehigh-affinity TGF-β receptors. The resulting cDNAs were inserted intothe vector pcDNA-1, which carries the CMV transcriptional promoter andthe SV40 origin of replication. The resulting library was then dividedinto pools of 10,000 independent recombinants each and DNA from eachpool was transfected into 1.5×10⁶ COS-7 cells grown on glass flaskettesby means of DEAE-dextran transfection procedure. Aruffo, A. and Seed,B., Proc. Natl. Acad. Sci. U.S.A. 84: 8573-8577 (1987); Gearing, D. etal., EMBO J. 8: 3667-3676 (1989); Mathews, L. S. and Vale, W. W., Cell65: 973-982 (1991). The transfected cells were cultured for 48-60 hoursand then exposed to radiolabelled TGF-β1 for four hours. Following thistreatment, the glass slides carrying these cells were washed extensivelyand fixed. These slides were dipped in liquid photographic emulsion andexamined by darkfield microscopy. While all of the receptor genes clonedto date by this procedure have undetectable or low constitutive levelsof expression in COS cells, we were hindered by the fact thatuntransfected COS cells already express substantial amounts of type IIITGF-β receptor. Such expression, estimated to be approximately 2×10⁵receptor molecules per cell, might well have generated an unacceptablyhigh level of background binding. However, since the detection procedureinvolves visualizing radiolabelled ligand-binding on individual cells,it was hoped that identifying occasional cells expressing substantiallyhigher levels of vector-encoded receptor would be possible. This hopewas vindicated in the initial experiments.

After screening nearly one million cDNA clones in this manner, a glassslide containing 20 positive transfectants was identified. The originalpool of expression constructs from which one such transfectant wasderived was split into 25 subpools of 1000 clones each and these weresubjected to a second round of screening. Two further rounds ofsib-selection resulted in the isolation of a cDNA clone (R3-OF) with a2.9 kb insert that induced high levels of TGF-β-binding proteins inapproximately 10% of COS cells into which it was transfected.

The specificity of this binding was validated by showing that additionof a 200-fold excess of unlabeled TGF-β competitor strongly reducedbinding of ¹²⁸ I-TGF-β to transfected cells. By taking into account atransfection efficiency of 10% and the high background of endogenousreceptor expression, we calculated that the level of total ¹²⁸ I-TGF-βbinding to each glass slide of cells transfected with this cDNA clonewas only 2-fold above the level seen with mock transfectants (data notshown). Nonetheless, this marginal increase in ligand-binding wassufficient to identify rare transfectants amidst a large field of cellsexpressing this background level of receptor.

The R3-OF cDNA encoded an open reading frame of 836 amino acid residuesof which the 3' most 18 were encoded by vector sequence, clearlyindicating that clone R3-OF was an incomplete cDNA insert which endedprematurely at the codon specifying alanine 818 (FIG. 4). R3-OF was usedas a probe to isolate a full-length cDNA from a rat 208 F lambda phagelibrary. This clone, termed R3-OFF, was 6 kb in length and encoded aprotein of 853 amino acids; its sequence was co-linear with that ofclone R3-OF.

Example 3 Characterization of the Product of the Full Length CloneR3-OFF

Characterization of the product of the full length clone R3-OFF wasundertaken in order to determine which of the three TGF-β receptors itspecified. To do so, COS transfectants were incubated withradioiodinated TGF-β, chemical crosslinker was added and the labelledreceptors were resolved by polyacrylamide gel electrophoresis.

Labelling of cell surface TGF-β receptors in this way resulted in thedetection of three distinct species on the surface of COS cells, asextensively by others (Massague, J. et al., Ann. NY Acad. Sci. 593:59-72 (1990). These included the two lower molecular weight type I andII receptors (65 and 85 kD) and the higher molecular weight type IIIproteoglycan, which migrated as a diffuse band of 280-330 kd. Enzymatictreatment of the proteoglycan with chondroitinase and heparitinaseyielded a core protein of approximately 100 kd. Binding to all threereceptor types was specific, in that it was completed by 200-fold excessof unlabeled TGF-β1.

Transfecting the R3-OFF cDNA caused a two-fold increase in expression ofthe type III receptor. When a cell lysate derived from COS cellstransfected with clone R3-OFF was treated with deglycosylating enzymes,the heterogenous 280-230 kd band was converted to a protein core whichco-migrated with the type III protein core seen in untransfected A10cells. Importantly, the recombinant protein core migrates differentlyfrom the endogenous COS cell type III protein core.

These observaltions were confirmed and extended in experiments usingstably transfected cells expressing the R3-OFF cDNA. L6 rat skeletonmuscle myoblasts normally do not express detectable type III mRNA orendogenous type III receptor (determined by radiolabelled ligand-bindingassay). Such cells were transfected with the isolated cDNA in the vectorpcDNA-neo. Cell clones stably expressing this clone in both the forwardand reverse orientations with respect to the CMV promoter were isolatedand analyzed by ligand-binding assay.

Introductior of either the full length clone R3-OFF or the partial cloneR3-OF in the forward orientation led to the de novo expression of thetype III receptor. L6 cells transfected with the cDNA in reversedorientation did not express this protein. The apparent size of theprotein core of the type III receptor in cells transfected with theR3-OF clone is smaller than that expressed by R3-OFF transfected cells,consistent with the difference in the sizes of the protein corespredicted from the respective nucleic acid sequences (FIGS. 1A-1C).

Unexpectedly the amount of radio-labelled ligand corss-linked to thetype II receptor is increased by 2.5 fold in cells expressing the typeIII cDNA, while the amount cross-linked to the type I receptor remainedunchanged. This apparent specific up-regulation of ligand-binding to thetype II receptor could be detected with all of the 15 stably transfectedL6 cell lines analyzed so far. This effect seems to be mediated by thetruncated clone R3-OF which lacks the cytoplasmic domain as well as bythe full-length clone R3-OFF.

Example 4 Expression of Type III Receptor

Northern blot analysis demonstrated that the type III receptor mRNA isexpressed as a single 6 kb message in several rat tissues. The level ofMRNA expression in the liver was less than in other tissues, a resultexpected from earlier surveys of various tissues using radioiodinatedTGF-μ1. Based on this information, it appears that clone R3-OFF, with a˜6 kb cDNA insert, represents a full length rat type III cDNA clone.

Cells of mouse origin (MEL and YH16) appear to express a smaller (˜5.5kb) message for the type III MRNA than those of pig, rat and humanorigin. In all of these cells, expression or absence of the type IIImRNA is consistent with the expression or absence of detectable cellsurface type III receptors with the notable exception of theretinoblastoma cell lines (Y79, Weri-1, Weri-24, and Weri-27). Thesecells have previously been shown to lack detectable surface expressionof type III receptor, a result confirmed by our own unpublished work. Itis striking that the type III receptor mRNA is expressed in these cellsat a level comparable to that of other cells that do indeed express typeIII receptor proteins at readily detectable levels. At this moment, wecan only conclude that TGF-β receptor III expression, which issubstantial in normal retinoblasts (AD12), has been down-regulated inthese retinoblastoma tumor cells, perhaps through post-transcriptionalmechanisms.

Example 5 Sequence Analysis of the Full-Length Type III cDNA

The full-length cDNA clone (R3-OFF), described in Example 4, wassubjected to sequence analysis. The full reading frame along withflanking sequences is presented in FIGS. 1A-1C. This reading frameencodes a protein of 853 amino acid residues, which is compatible withthe 100 kD observed for the fully deglycosylated TGF-β type IIIreceptor.

Two segments of derived protein sequence (underlined and italicized,residues 378-388 and 427-434) precisely match those determined earlierfrom direct biochemical analysis of the purified receptor protein. Thisfurther secured the identity of this isolated cDNA clone as encoding therat type III receptor.

This TGF-β binding protein has an unusual structure for a cytokinereceptor. Hydropathy analysis indicates a N-terminal signal sequence,followed by a long, hydrophilic N-terminal region (Kyte, J. and R. F.Doolittle, J. Mol. Biol. 157: 105-132 (1982)). A 27 residue region ofstrong hydrophobicity (underlined, residues 786-812) toward theC-terminus represents the single putative transmembrane domain. Thissuggests that nearly all of the receptor is composed of an N-terminalextracellular domain that is anchored to the plasma membrane near itsC-terminus. A relatively short C-terminal tail of 41 residues representsthe putative cytoplasmic domain.

The clone R3-OF was also analyzed and found to be a truncated version ofR3-OFF, with an identical open reading frame but whose last encodedresidue is alanine 818 (FIGS. 1A-1C).

In R3-OFF there are six consensus N-linked glycosylation sites and 15cysteines (indicated in FIGS. 1A-1C). There, is at least one consensusglycosaminoglycan addition site at serine 535 (Bernfield, M. and K. C.Hooper, Ann. N.Y. Acad. Sci. in press (1991), and numerous Ser-Glyresidues that are potential sites for GAG conjugation. A consensusprotein kinase C site is also present at residue 817.

Only one other gene described to date, a glycoprotein expressed in highquantities by endothelial cells and termed endoglin (Gougos and Letarte,1990), contains a related amino acid sequence. Overall, there is ˜30%identity with the type III receptor over the entire 645 amino acidresidue length of endoglin. The most homologous regions between thesequences of the type III receptor and endoglin (74% identity) fallsprimarily in the putative transmembrane and cytoplasmic domains. Similarto the general structure of type III receptor, endoglin is aglycoprotein which contains a large hydrophilic and presumablyextracellular N-terminal domain followed by a putative transmembranedomain and a short cytoplasmic tail of 47 amino acid residues. Thebiological role of endoglin is unclear, though it has been suggestedthat it may involve cell-cell recognition through interactions of an"RGD" sequence on its ectodomain with other adhesion molecules. Unlikethe TGF-β type III receptor, endoglin does not carry GAG groups.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingnot more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 8                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Ile Leu Leu Asp Pro Asp His Pro Pro Ala Le - #u                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Gln Ala Pro Phe Pro Ile Asn Phe Met Ile Al - #a                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Gln Pro Ile Val Pro Ser Val Gln                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Phe Tyr Val Glu Gln Gly Tyr Gly Arg                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3237 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 241..2799                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - CAGGAGGTGA AAGTCCCCGG CGGTCCGGAT GGCGCAGTTG CACTGCGCTG CT - #GAGCTCGC         60                                                                          - GGCCGCCTGC GCACACTGGG GGGACTCGCT TCGGCTAGTA ACTCCTCCAC CT - #CGCGGCGG        120                                                                          - ACGACCGGTC CTGGACACGC TGCCTGCGAG GCAAGTTGAA CAGTGCAGAG AA - #GGATCTTA        180                                                                          - AAGCTACACC CGACTTGCCA CGATTGCCTT CAATCTGAAG AACCAAAGGC TG - #TTGGAGAG        240                                                                          - ATG GCA GTG ACA TCC CAC CAC ATG ATC CCG GT - #G ATG GTT GTC CTG ATG          288                                                                          Met Ala Val Thr Ser His His Met Ile Pro Va - #l Met Val Val Leu Met           #                 15                                                          - AGC GCC TGC CTG GCC ACC GCC GGT CCA GAG CC - #C AGC ACC CGG TGT GAA          336                                                                          Ser Ala Cys Leu Ala Thr Ala Gly Pro Glu Pr - #o Ser Thr Arg Cys Glu           #             30                                                              - CTG TCA CCA ATC AAC GCC TCT CAC CCA GTC CA - #G GCC TTG ATG GAG AGC          384                                                                          Leu Ser Pro Ile Asn Ala Ser His Pro Val Gl - #n Ala Leu Met Glu Ser           #         45                                                                  - TTC ACC GTT CTG TCT GGC TGT GCC AGC AGA GG - #C ACC ACC GGG CTG CCA          432                                                                          Phe Thr Val Leu Ser Gly Cys Ala Ser Arg Gl - #y Thr Thr Gly Leu Pro           #     60                                                                      - AGG GAG GTC CAT GTC CTA AAC CTC CGA AGT AC - #A GAT CAG GGA CCA GGC          480                                                                          Arg Glu Val His Val Leu Asn Leu Arg Ser Th - #r Asp Gln Gly Pro Gly           # 80                                                                          - CAG CGG CAG AGA GAG GTT ACC CTG CAC CTG AA - #C CCC ATT GCC TCG GTG          528                                                                          Gln Arg Gln Arg Glu Val Thr Leu His Leu As - #n Pro Ile Ala Ser Val           #                 95                                                          - CAC ACT CAC CAC AAA CCT ATC GTG TTC CTG CT - #C AAC TCC CCC CAG CCC          576                                                                          His Thr His His Lys Pro Ile Val Phe Leu Le - #u Asn Ser Pro Gln Pro           #           110                                                               - CTG GTG TGG CAT CTG AAG ACG GAG AGA CTG GC - #C GCT GGT GTC CCC AGA          624                                                                          Leu Val Trp His Leu Lys Thr Glu Arg Leu Al - #a Ala Gly Val Pro Arg           #       125                                                                   - CTC TTC CTG GTT TCG GAG GGT TCT GTG GTC CA - #G TTT CCA TCA GGA AAC          672                                                                          Leu Phe Leu Val Ser Glu Gly Ser Val Val Gl - #n Phe Pro Ser Gly Asn           #   140                                                                       - TTC TCC TTG ACA GCA GAA ACA GAG GAA AGG AA - #T TTC CCT CAA GAA AAT          720                                                                          Phe Ser Leu Thr Ala Glu Thr Glu Glu Arg As - #n Phe Pro Gln Glu Asn           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - GAA CAT CTC GTG CGC TGG GCC CAA AAG GAA TA - #T GGA GCA GTG ACT TCG          768                                                                          Glu His Leu Val Arg Trp Ala Gln Lys Glu Ty - #r Gly Ala Val Thr Ser           #               175                                                           - TTC ACT GAA CTC AAG ATA GCA AGA AAC ATC TA - #T ATT AAA GTG GGA GAA          816                                                                          Phe Thr Glu Leu Lys Ile Ala Arg Asn Ile Ty - #r Ile Lys Val Gly Glu           #           190                                                               - GAT CAA GTG TTT CCT CCT ACG TGT AAC ATA GG - #G AAG AAT TTC CTC TCA          864                                                                          Asp Gln Val Phe Pro Pro Thr Cys Asn Ile Gl - #y Lys Asn Phe Leu Ser           #       205                                                                   - CTC AAT TAC CTT GCC GAG TAC CTT CAA CCC AA - #A GCC GCC GAA GGT TGT          912                                                                          Leu Asn Tyr Leu Ala Glu Tyr Leu Gln Pro Ly - #s Ala Ala Glu Gly Cys           #   220                                                                       - GTC CTG CCC AGT CAG CCC CAT GAA AAG GAA GT - #A CAC ATC ATC GAG TTA          960                                                                          Val Leu Pro Ser Gln Pro His Glu Lys Glu Va - #l His Ile Ile Glu Leu           225                 2 - #30                 2 - #35                 2 -       #40                                                                           - ATT ACC CCC AGC TCG AAC CCT TAC AGC GCT TT - #C CAG GTG GAT ATA ATA         1008                                                                          Ile Thr Pro Ser Ser Asn Pro Tyr Ser Ala Ph - #e Gln Val Asp Ile Ile           #               255                                                           - GTT GAC ATA CGA CCT GCT CAA GAG GAT CCC GA - #G GTG GTC AAA AAC CTT         1056                                                                          Val Asp Ile Arg Pro Ala Gln Glu Asp Pro Gl - #u Val Val Lys Asn Leu           #           270                                                               - GTC CTG ATC TTG AAG TGC AAA AAG TCT GTC AA - #C TGG GTG ATC AAG TCT         1104                                                                          Val Leu Ile Leu Lys Cys Lys Lys Ser Val As - #n Trp Val Ile Lys Ser           #       285                                                                   - TTT GAC GTC AAG GGA AAC TTG AAA GTC ATT GC - #T CCC AAC AGT ATC GGC         1152                                                                          Phe Asp Val Lys Gly Asn Leu Lys Val Ile Al - #a Pro Asn Ser Ile Gly           #   300                                                                       - TTT GGA AAA GAG AGT GAA CGA TCC ATG ACA AT - #G ACC AAA TTG GTA AGA         1200                                                                          Phe Gly Lys Glu Ser Glu Arg Ser Met Thr Me - #t Thr Lys Leu Val Arg           305                 3 - #10                 3 - #15                 3 -       #20                                                                           - GAT GAC ATC CCT TCC ACC CAA GAG AAT CTG AT - #G AAG TGG GCA CTG GAC         1248                                                                          Asp Asp Ile Pro Ser Thr Gln Glu Asn Leu Me - #t Lys Trp Ala Leu Asp           #               335                                                           - AAT GGC TAC AGG CCA GTG ACG TCA TAC ACA AT - #G GCT CCC GTG GCT AAT         1296                                                                          Asn Gly Tyr Arg Pro Val Thr Ser Tyr Thr Me - #t Ala Pro Val Ala Asn           #           350                                                               - AGA TTT CAT CTT CGG CTT GAG AAC AAC GAG GA - #G ATG AGA GAT GAG GAA         1344                                                                          Arg Phe His Leu Arg Leu Glu Asn Asn Glu Gl - #u Met Arg Asp Glu Glu           #       365                                                                   - GTC CAC ACC ATT CCT CCT GAG CTT CGT ATC CT - #G CTG GAC CCT GAC CAC         1392                                                                          Val His Thr Ile Pro Pro Glu Leu Arg Ile Le - #u Leu Asp Pro Asp His           #   380                                                                       - CCG CCC GCC CTG GAC AAC CCA CTC TTC CCA GG - #A GAG GGA AGC CCA AAT         1440                                                                          Pro Pro Ala Leu Asp Asn Pro Leu Phe Pro Gl - #y Glu Gly Ser Pro Asn           385                 3 - #90                 3 - #95                 4 -       #00                                                                           - GGT GGT CTC CCC TTT CCA TTC CCG GAT ATC CC - #C AGG AGA GGC TGG AAG         1488                                                                          Gly Gly Leu Pro Phe Pro Phe Pro Asp Ile Pr - #o Arg Arg Gly Trp Lys           #               415                                                           - GAG GGC GAA GAT AGG ATC CCC CGG CCA AAG CA - #G CCC ATC GTT CCC AGT         1536                                                                          Glu Gly Glu Asp Arg Ile Pro Arg Pro Lys Gl - #n Pro Ile Val Pro Ser           #           430                                                               - GTT CAA CTG CTT CCT GAC CAC CGA GAA CCA GA - #A GAA GTG CAA GGG GGC         1584                                                                          Val Gln Leu Leu Pro Asp His Arg Glu Pro Gl - #u Glu Val Gln Gly Gly           #       445                                                                   - GTG GAC ATC GCC CTG TCA GTC AAA TGT GAC CA - #T GAA AAG ATG GTC GTG         1632                                                                          Val Asp Ile Ala Leu Ser Val Lys Cys Asp Hi - #s Glu Lys Met Val Val           #   460                                                                       - GCT GTA GAC AAA GAC TCT TTC CAG ACC AAT GG - #C TAC TCA GGG ATG GAG         1680                                                                          Ala Val Asp Lys Asp Ser Phe Gln Thr Asn Gl - #y Tyr Ser Gly Met Glu           465                 4 - #70                 4 - #75                 4 -       #80                                                                           - CTC ACC CTG TTG GAT CCT TCG TGT AAA GCC AA - #A ATG AAT GGT ACT CAC         1728                                                                          Leu Thr Leu Leu Asp Pro Ser Cys Lys Ala Ly - #s Met Asn Gly Thr His           #               495                                                           - TTT GTT CTC GAG TCT CCC CTG AAT GGC TGT GG - #T ACT CGA CAT CGG AGG         1776                                                                          Phe Val Leu Glu Ser Pro Leu Asn Gly Cys Gl - #y Thr Arg His Arg Arg           #           510                                                               - TCG ACC CCG GAT GGT GTG GTT TAC TAT AAC TC - #T ATT GTG GTG CAG GCT         1824                                                                          Ser Thr Pro Asp Gly Val Val Tyr Tyr Asn Se - #r Ile Val Val Gln Ala           #       525                                                                   - CCG TCC CCT GGG GAT AGC AGT GGC TGG CCT GA - #T GGC TAT GAA GAC TTG         1872                                                                          Pro Ser Pro Gly Asp Ser Ser Gly Trp Pro As - #p Gly Tyr Glu Asp Leu           #   540                                                                       - GAG TCA GGC GAT AAT GGA TTT CCT GGA GAC GG - #G GAT GAA GGA GAA ACT         1920                                                                          Glu Ser Gly Asp Asn Gly Phe Pro Gly Asp Gl - #y Asp Glu Gly Glu Thr           545                 5 - #50                 5 - #55                 5 -       #60                                                                           - GCC CCC CTG AGC CGA GCT GGA GTG GTG GTG TT - #T AAC TGC AGC TTG CGG         1968                                                                          Ala Pro Leu Ser Arg Ala Gly Val Val Val Ph - #e Asn Cys Ser Leu Arg           #               575                                                           - CAG CTG AGG AAT CCC AGT GGC TTC CAG GGC CA - #G CTC GAT GGA AAT GCT         2016                                                                          Gln Leu Arg Asn Pro Ser Gly Phe Gln Gly Gl - #n Leu Asp Gly Asn Ala           #           590                                                               - ACC TTC AAC ATG GAG CTG TAT AAC ACA GAC CT - #C TTT CTG GTG CCC TCC         2064                                                                          Thr Phe Asn Met Glu Leu Tyr Asn Thr Asp Le - #u Phe Leu Val Pro Ser           #       605                                                                   - CCA GGG GTC TTC TCT GTG GCA GAG AAC GAG CA - #T GTT TAT GTT GAG GTG         2112                                                                          Pro Gly Val Phe Ser Val Ala Glu Asn Glu Hi - #s Val Tyr Val Glu Val           #   620                                                                       - TCT GTC ACC AAG GCT GAC CAA GAT CTG GGA TT - #C GCC ATC CAA ACC TGC         2160                                                                          Ser Val Thr Lys Ala Asp Gln Asp Leu Gly Ph - #e Ala Ile Gln Thr Cys           625                 6 - #30                 6 - #35                 6 -       #40                                                                           - TTT CTC TCT CCA TAC TCC AAC CCA GAC AGA AT - #G TCT GAT TAC ACC ATC         2208                                                                          Phe Leu Ser Pro Tyr Ser Asn Pro Asp Arg Me - #t Ser Asp Tyr Thr Ile           #               655                                                           - ATC GAG AAC ATC TGT CCG AAA GAC GAC TCT GT - #G AAG TTC TAC AGC TCC         2256                                                                          Ile Glu Asn Ile Cys Pro Lys Asp Asp Ser Va - #l Lys Phe Tyr Ser Ser           #           670                                                               - AAG AGA GTG CAC TTT CCC ATC CCG CAT GCT GA - #G GTG GAC AAG AAG CGC         2304                                                                          Lys Arg Val His Phe Pro Ile Pro His Ala Gl - #u Val Asp Lys Lys Arg           #       685                                                                   - TTC AGC TTC CTG TTC AAG TCT GTG TTC AAC AC - #C TCC CTG CTC TTC CTG         2352                                                                          Phe Ser Phe Leu Phe Lys Ser Val Phe Asn Th - #r Ser Leu Leu Phe Leu           #   700                                                                       - CAC TGC GAG TTG ACT CTG TGC TCC AGG AAG AA - #G GGC TCC CTG AAG CTG         2400                                                                          His Cys Glu Leu Thr Leu Cys Ser Arg Lys Ly - #s Gly Ser Leu Lys Leu           705                 7 - #10                 7 - #15                 7 -       #20                                                                           - CCG AGG TGT GTG ACT CCT GAC GAC GCC TGC AC - #T TCT CTC GAT GCC ACC         2448                                                                          Pro Arg Cys Val Thr Pro Asp Asp Ala Cys Th - #r Ser Leu Asp Ala Thr           #               735                                                           - ATG ATC TGG ACC ATG ATG CAG AAT AAG AAG AC - #A TTC ACC AAG CCC CTG         2496                                                                          Met Ile Trp Thr Met Met Gln Asn Lys Lys Th - #r Phe Thr Lys Pro Leu           #           750                                                               - GCT GTG GTC CTC CAG GTA GAC TAT AAA GAA AA - #T GTT CCC AGC ACT AAG         2544                                                                          Ala Val Val Leu Gln Val Asp Tyr Lys Glu As - #n Val Pro Ser Thr Lys           #       765                                                                   - GAT TCC AGT CCA ATT CCT CCT CCT CCT CCA CA - #G ATT TTC CAT GGC CTG         2592                                                                          Asp Ser Ser Pro Ile Pro Pro Pro Pro Pro Gl - #n Ile Phe His Gly Leu           #   780                                                                       - GAC ACG CTC ACC GTG ATG GGC ATT GCA TTT GC - #A GCA TTT GTG ATC GGA         2640                                                                          Asp Thr Leu Thr Val Met Gly Ile Ala Phe Al - #a Ala Phe Val Ile Gly           785                 7 - #90                 7 - #95                 8 -       #00                                                                           - GCG CTC CTG ACG GGG GCC TTG TGG TAC ATC TA - #C TCC CAC ACA GGG GAG         2688                                                                          Ala Leu Leu Thr Gly Ala Leu Trp Tyr Ile Ty - #r Ser His Thr Gly Glu           #               815                                                           - ACA GCA CGA AGG CAG CAA GTC CCT ACC TCG CC - #G CCA GCC TCG GAG AAC         2736                                                                          Thr Ala Arg Arg Gln Gln Val Pro Thr Ser Pr - #o Pro Ala Ser Glu Asn           #           830                                                               - AGC AGC GCG GCC CAC AGC ATC GGC AGC ACT CA - #G AGT ACC CCC TGC TCT         2784                                                                          Ser Ser Ala Ala His Ser Ile Gly Ser Thr Gl - #n Ser Thr Pro Cys Ser           #       845                                                                   - AGC AGC AGC ACA GCC TAGGTGGACA GACAGACGCC CGCCCACCG - #C AGCCAGGGCA         2839                                                                          Ser Ser Ser Thr Ala                                                               850                                                                       - GGGCCCGATG CCAGTGCTGC GTGTCCACAG TCAGAAGTCT TGATCTGGGC TC - #CCTGTAAA       2899                                                                          - GAAAGAGTGA ATTTCAGTAT ACAGACAGCC AGTTCTACCC ACCCCTTACC AC - #GGCCCACA       2959                                                                          - TAAATGTGAC CCTGGGCATC TGTCACACGA AAGCTAAGCT GGTGGCCTTC CC - #CACCAGCC       3019                                                                          - CCTCGCAGGA TGGGGGTTTC AATGTGAAAC ATCTGCCAGT TTTGTTTTGT TT - #TTTTAATG       3079                                                                          - CTGCTTTGTC CAGGTGTCCA AACATCCATC ATTTGGGGTG GTCTGTTTTA CA - #GAGTAAAG       3139                                                                          - GAGGCGGTGA AGGGACGTCA GCTAGTGTGT AGAGCCAAGG GGAGACAGCT AG - #GATTCTCG       3199                                                                          #   3237           TGTA AAATAGAAGA CACGCTCC                                   - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 853 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Met Ala Val Thr Ser His His Met Ile Pro Va - #l Met Val Val Leu Met         #                 15                                                          - Ser Ala Cys Leu Ala Thr Ala Gly Pro Glu Pr - #o Ser Thr Arg Cys Glu         #             30                                                              - Leu Ser Pro Ile Asn Ala Ser His Pro Val Gl - #n Ala Leu Met Glu Ser         #         45                                                                  - Phe Thr Val Leu Ser Gly Cys Ala Ser Arg Gl - #y Thr Thr Gly Leu Pro         #     60                                                                      - Arg Glu Val His Val Leu Asn Leu Arg Ser Th - #r Asp Gln Gly Pro Gly         # 80                                                                          - Gln Arg Gln Arg Glu Val Thr Leu His Leu As - #n Pro Ile Ala Ser Val         #                 95                                                          - His Thr His His Lys Pro Ile Val Phe Leu Le - #u Asn Ser Pro Gln Pro         #           110                                                               - Leu Val Trp His Leu Lys Thr Glu Arg Leu Al - #a Ala Gly Val Pro Arg         #       125                                                                   - Leu Phe Leu Val Ser Glu Gly Ser Val Val Gl - #n Phe Pro Ser Gly Asn         #   140                                                                       - Phe Ser Leu Thr Ala Glu Thr Glu Glu Arg As - #n Phe Pro Gln Glu Asn         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Glu His Leu Val Arg Trp Ala Gln Lys Glu Ty - #r Gly Ala Val Thr Ser         #               175                                                           - Phe Thr Glu Leu Lys Ile Ala Arg Asn Ile Ty - #r Ile Lys Val Gly Glu         #           190                                                               - Asp Gln Val Phe Pro Pro Thr Cys Asn Ile Gl - #y Lys Asn Phe Leu Ser         #       205                                                                   - Leu Asn Tyr Leu Ala Glu Tyr Leu Gln Pro Ly - #s Ala Ala Glu Gly Cys         #   220                                                                       - Val Leu Pro Ser Gln Pro His Glu Lys Glu Va - #l His Ile Ile Glu Leu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ile Thr Pro Ser Ser Asn Pro Tyr Ser Ala Ph - #e Gln Val Asp Ile Ile         #               255                                                           - Val Asp Ile Arg Pro Ala Gln Glu Asp Pro Gl - #u Val Val Lys Asn Leu         #           270                                                               - Val Leu Ile Leu Lys Cys Lys Lys Ser Val As - #n Trp Val Ile Lys Ser         #       285                                                                   - Phe Asp Val Lys Gly Asn Leu Lys Val Ile Al - #a Pro Asn Ser Ile Gly         #   300                                                                       - Phe Gly Lys Glu Ser Glu Arg Ser Met Thr Me - #t Thr Lys Leu Val Arg         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Asp Asp Ile Pro Ser Thr Gln Glu Asn Leu Me - #t Lys Trp Ala Leu Asp         #               335                                                           - Asn Gly Tyr Arg Pro Val Thr Ser Tyr Thr Me - #t Ala Pro Val Ala Asn         #           350                                                               - Arg Phe His Leu Arg Leu Glu Asn Asn Glu Gl - #u Met Arg Asp Glu Glu         #       365                                                                   - Val His Thr Ile Pro Pro Glu Leu Arg Ile Le - #u Leu Asp Pro Asp His         #   380                                                                       - Pro Pro Ala Leu Asp Asn Pro Leu Phe Pro Gl - #y Glu Gly Ser Pro Asn         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Gly Gly Leu Pro Phe Pro Phe Pro Asp Ile Pr - #o Arg Arg Gly Trp Lys         #               415                                                           - Glu Gly Glu Asp Arg Ile Pro Arg Pro Lys Gl - #n Pro Ile Val Pro Ser         #           430                                                               - Val Gln Leu Leu Pro Asp His Arg Glu Pro Gl - #u Glu Val Gln Gly Gly         #       445                                                                   - Val Asp Ile Ala Leu Ser Val Lys Cys Asp Hi - #s Glu Lys Met Val Val         #   460                                                                       - Ala Val Asp Lys Asp Ser Phe Gln Thr Asn Gl - #y Tyr Ser Gly Met Glu         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Leu Thr Leu Leu Asp Pro Ser Cys Lys Ala Ly - #s Met Asn Gly Thr His         #               495                                                           - Phe Val Leu Glu Ser Pro Leu Asn Gly Cys Gl - #y Thr Arg His Arg Arg         #           510                                                               - Ser Thr Pro Asp Gly Val Val Tyr Tyr Asn Se - #r Ile Val Val Gln Ala         #       525                                                                   - Pro Ser Pro Gly Asp Ser Ser Gly Trp Pro As - #p Gly Tyr Glu Asp Leu         #   540                                                                       - Glu Ser Gly Asp Asn Gly Phe Pro Gly Asp Gl - #y Asp Glu Gly Glu Thr         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Ala Pro Leu Ser Arg Ala Gly Val Val Val Ph - #e Asn Cys Ser Leu Arg         #               575                                                           - Gln Leu Arg Asn Pro Ser Gly Phe Gln Gly Gl - #n Leu Asp Gly Asn Ala         #           590                                                               - Thr Phe Asn Met Glu Leu Tyr Asn Thr Asp Le - #u Phe Leu Val Pro Ser         #       605                                                                   - Pro Gly Val Phe Ser Val Ala Glu Asn Glu Hi - #s Val Tyr Val Glu Val         #   620                                                                       - Ser Val Thr Lys Ala Asp Gln Asp Leu Gly Ph - #e Ala Ile Gln Thr Cys         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Phe Leu Ser Pro Tyr Ser Asn Pro Asp Arg Me - #t Ser Asp Tyr Thr Ile         #               655                                                           - Ile Glu Asn Ile Cys Pro Lys Asp Asp Ser Va - #l Lys Phe Tyr Ser Ser         #           670                                                               - Lys Arg Val His Phe Pro Ile Pro His Ala Gl - #u Val Asp Lys Lys Arg         #       685                                                                   - Phe Ser Phe Leu Phe Lys Ser Val Phe Asn Th - #r Ser Leu Leu Phe Leu         #   700                                                                       - His Cys Glu Leu Thr Leu Cys Ser Arg Lys Ly - #s Gly Ser Leu Lys Leu         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Pro Arg Cys Val Thr Pro Asp Asp Ala Cys Th - #r Ser Leu Asp Ala Thr         #               735                                                           - Met Ile Trp Thr Met Met Gln Asn Lys Lys Th - #r Phe Thr Lys Pro Leu         #           750                                                               - Ala Val Val Leu Gln Val Asp Tyr Lys Glu As - #n Val Pro Ser Thr Lys         #       765                                                                   - Asp Ser Ser Pro Ile Pro Pro Pro Pro Pro Gl - #n Ile Phe His Gly Leu         #   780                                                                       - Asp Thr Leu Thr Val Met Gly Ile Ala Phe Al - #a Ala Phe Val Ile Gly         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Ala Leu Leu Thr Gly Ala Leu Trp Tyr Ile Ty - #r Ser His Thr Gly Glu         #               815                                                           - Thr Ala Arg Arg Gln Gln Val Pro Thr Ser Pr - #o Pro Ala Ser Glu Asn         #           830                                                               - Ser Ser Ala Ala His Ser Ile Gly Ser Thr Gl - #n Ser Thr Pro Cys Ser         #       845                                                                   - Ser Ser Ser Thr Ala                                                             850                                                                       - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2090 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 336..2038                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - GTTGGCGAGG AGTTTCCTGT TTCCCCCGCA GCGCTGAGTT GAAGTTGAGT GA - #GTCACTCG         60                                                                          - CGCGCACGGA GCGACGACAC CCCCGCGCGT GCACCCGCTC GGGACAGGAG CC - #GGACTCCT        120                                                                          - GTGCAGCTTC CCTCGGCCGC CGGGGGCCTC CCCGCGCCTC GCCGGCCTCC AG - #GCCCCTCC        180                                                                          - TGGCTGGCGA GCGGGCGCCA CATCTGGCCC GCACATCTGC GCTGCCGGCC CG - #GCGCGGGG        240                                                                          - TCCGGAGAGG GCGCGGCGCG GAGCGCAGCC AGGGGTCCGG GAAGGCGCCG TC - #CGTGCGCT        300                                                                          #GGG CTG CTC       353A GCAGCGGGGT CTGCC ATG GGT CGG                          #   Met Gly Arg Gly Leu Leu                                                   #  5  1                                                                       - AGG GGC CTG TGG CCG CTG CAC ATC GTC CTG TG - #G ACG CGT ATC GCC AGC          401                                                                          Arg Gly Leu Trp Pro Leu His Ile Val Leu Tr - #p Thr Arg Ile Ala Ser           #             20                                                              - ACG ATC CCA CCG CAC GTT CAG AAG TCG GTT AA - #T AAC GAC ATG ATA GTC          449                                                                          Thr Ile Pro Pro His Val Gln Lys Ser Val As - #n Asn Asp Met Ile Val           #         35                                                                  - ACT GAC AAC AAC GGT GCA GTC AAG TTT CCA CA - #A CTG TGT AAA TTT TGT          497                                                                          Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gl - #n Leu Cys Lys Phe Cys           #     50                                                                      - GAT GTG AGA TTT TCC ACC TGT GAC AAC CAG AA - #A TCC TGC ATG AGC AAC          545                                                                          Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Ly - #s Ser Cys Met Ser Asn           # 70                                                                          - TGC AGC ATC ACC TCC ATC TGT GAG AAG CCA CA - #G GAA GTC TGT GTG GCT          593                                                                          Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gl - #n Glu Val Cys Val Ala           #                 85                                                          - GTA TGG AGA AAG AAT GAC GAG AAC ATA ACA CT - #A GAG ACA GTT TGC CAT          641                                                                          Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Le - #u Glu Thr Val Cys His           #            100                                                              - GAC CCC AAG CTC CCC TAC CAT GAC TTT ATT CT - #G GAA GAT GCT GCT TCT          689                                                                          Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Le - #u Glu Asp Ala Ala Ser           #       115                                                                   - CCA AAG TGC ATT ATG AAG GAA AAA AAA AAG CC - #T GGT GAG ACT TTC TTC          737                                                                          Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pr - #o Gly Glu Thr Phe Phe           #   130                                                                       - ATG TGT TCC TGT AGC TCT GAT GAG TGC AAT GA - #C AAC ATC ATC TTC TCA          785                                                                          Met Cys Ser Cys Ser Ser Asp Glu Cys Asn As - #p Asn Ile Ile Phe Ser           135                 1 - #40                 1 - #45                 1 -       #50                                                                           - GAA GAA TAT AAC ACC AGC AAT CCT GAC TTG TT - #G CTA GTC ATA TTT CAA          833                                                                          Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu Le - #u Leu Val Ile Phe Gln           #               165                                                           - GTG ACA GGC ATC AGC CTC CTG CCA CCA CTG GG - #A GTT GCC ATA TCT GTC          881                                                                          Val Thr Gly Ile Ser Leu Leu Pro Pro Leu Gl - #y Val Ala Ile Ser Val           #           180                                                               - ATC ATC ATC TTC TAC TGC TAC CGC GTT AAC CG - #G CAG CAG AAG CTG AGT          929                                                                          Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn Ar - #g Gln Gln Lys Leu Ser           #       195                                                                   - TCA ACC TGG GAA ACC GGC AAG ACG CGG AAG CT - #C ATG GAG TTC AGC GAG          977                                                                          Ser Thr Trp Glu Thr Gly Lys Thr Arg Lys Le - #u Met Glu Phe Ser Glu           #   210                                                                       - CAC TGT GCC ATC ATC CTG GAA GAT GAC CGC TC - #T GAC ATC AGC TCC ACG         1025                                                                          His Cys Ala Ile Ile Leu Glu Asp Asp Arg Se - #r Asp Ile Ser Ser Thr           215                 2 - #20                 2 - #25                 2 -       #30                                                                           - TGT GCC AAC AAC ATC AAC CAC AAC ACA GAG CT - #G CTG CCC ATT GAG CTG         1073                                                                          Cys Ala Asn Asn Ile Asn His Asn Thr Glu Le - #u Leu Pro Ile Glu Leu           #               245                                                           - GAC ACC CTG GTG GGG AAA GGT CGC TTT GCT GA - #G GTC TAT AAG GCC AAG         1121                                                                          Asp Thr Leu Val Gly Lys Gly Arg Phe Ala Gl - #u Val Tyr Lys Ala Lys           #           260                                                               - CTG AAG CAG AAC ACT TCA GAG CAG TTT GAG AC - #A GTG GCA GTC AAG ATC         1169                                                                          Leu Lys Gln Asn Thr Ser Glu Gln Phe Glu Th - #r Val Ala Val Lys Ile           #       275                                                                   - TTT CCC TAT GAG GAG TAT GCC TCT TGG AAG AC - #A GAG AAG GAC ATC TTC         1217                                                                          Phe Pro Tyr Glu Glu Tyr Ala Ser Trp Lys Th - #r Glu Lys Asp Ile Phe           #   290                                                                       - TCA GAC ATC AAT CTG AAG CAT GAG AAC ATA CT - #C CAG TTC CTG ACG GCT         1265                                                                          Ser Asp Ile Asn Leu Lys His Glu Asn Ile Le - #u Gln Phe Leu Thr Ala           295                 3 - #00                 3 - #05                 3 -       #10                                                                           - GAG GAG CGG AAG ACG GAG TTG GGG AAA CAA TA - #C TGG CTG ATC ACC GCC         1313                                                                          Glu Glu Arg Lys Thr Glu Leu Gly Lys Gln Ty - #r Trp Leu Ile Thr Ala           #               325                                                           - TTC CAC GCC AAG GGC AAC CTA CAG GAG TAC CT - #G ACG CGG CAT GTC ATC         1361                                                                          Phe His Ala Lys Gly Asn Leu Gln Glu Tyr Le - #u Thr Arg His Val Ile           #           340                                                               - AGC TGG GAG GAC CTG CGC AAG CTG GGC AGC TC - #C CTC GCC CGG GGG ATT         1409                                                                          Ser Trp Glu Asp Leu Arg Lys Leu Gly Ser Se - #r Leu Ala Arg Gly Ile           #       355                                                                   - GCT CAC CTC CAC AGT GAT CAC ACT CCA TGT GG - #G AGG CCC AAG ATG CCC         1457                                                                          Ala His Leu His Ser Asp His Thr Pro Cys Gl - #y Arg Pro Lys Met Pro           #   370                                                                       - ATC GTG CAC AGG GAC CTC AAG AGC TCC AAT AT - #C CTC GTG AAG AAC GAC         1505                                                                          Ile Val His Arg Asp Leu Lys Ser Ser Asn Il - #e Leu Val Lys Asn Asp           375                 3 - #80                 3 - #85                 3 -       #90                                                                           - CTA ACC TGC TGC CTG TGT GAC TTT GGG CTT TC - #C CTG CGT CTG GAC CCT         1553                                                                          Leu Thr Cys Cys Leu Cys Asp Phe Gly Leu Se - #r Leu Arg Leu Asp Pro           #               405                                                           - ACT CTG TCT GTG GAT GAC CTG GCT AAC AGT GG - #G CAG GTG GGA ACT GCA         1601                                                                          Thr Leu Ser Val Asp Asp Leu Ala Asn Ser Gl - #y Gln Val Gly Thr Ala           #           420                                                               - AGA TAC ATG GCT CCA GAA GTC CTA GAA TCC AG - #G ATG AAT TTG GAG AAT         1649                                                                          Arg Tyr Met Ala Pro Glu Val Leu Glu Ser Ar - #g Met Asn Leu Glu Asn           #       435                                                                   - GCT GAG TCC TTC AAG CAG ACC GAT GTC TAC TC - #C ATG GCT CTG GTG CTC         1697                                                                          Ala Glu Ser Phe Lys Gln Thr Asp Val Tyr Se - #r Met Ala Leu Val Leu           #   450                                                                       - TGG GAA ATG ACA TCT CGC TGT AAT GCA GTG GG - #A GAA GTA AAA GAT TAT         1745                                                                          Trp Glu Met Thr Ser Arg Cys Asn Ala Val Gl - #y Glu Val Lys Asp Tyr           455                 4 - #60                 4 - #65                 4 -       #70                                                                           - GAG CCT CCA TTT GGT TCC AAG GTG CGG GAG CA - #C CCC TGT GTC GAA AGC         1793                                                                          Glu Pro Pro Phe Gly Ser Lys Val Arg Glu Hi - #s Pro Cys Val Glu Ser           #               485                                                           - ATG AAG GAC AAC GTG TTG AGA GAT CGA GGG CG - #A CCA GAA ATT CCC AGC         1841                                                                          Met Lys Asp Asn Val Leu Arg Asp Arg Gly Ar - #g Pro Glu Ile Pro Ser           #           500                                                               - TTC TGG CTC AAC CAC CAG GGC ATC CAG ATG GT - #G TGT GAG ACG TTG ACT         1889                                                                          Phe Trp Leu Asn His Gln Gly Ile Gln Met Va - #l Cys Glu Thr Leu Thr           #       515                                                                   - GAG TGC TGG GAC CAC GAC CCA GAG GCC CGT CT - #C ACA GCC CAG TGT GTG         1937                                                                          Glu Cys Trp Asp His Asp Pro Glu Ala Arg Le - #u Thr Ala Gln Cys Val           #   530                                                                       - GCA GAA CGC TTC AGT GAG CTG GAG CAT CTG GA - #C AGG CTC TCG GGG AGG         1985                                                                          Ala Glu Arg Phe Ser Glu Leu Glu His Leu As - #p Arg Leu Ser Gly Arg           535                 5 - #40                 5 - #45                 5 -       #50                                                                           - AGC TGC TCG GAG GAG AAG ATT CCT GAA GAC GG - #C TCC CTA AAC ACT ACC         2033                                                                          Ser Cys Ser Glu Glu Lys Ile Pro Glu Asp Gl - #y Ser Leu Asn Thr Thr           #               565                                                           - AAA TA GCTCTTATGG GGCAGGCTGG GCATGTCCAA AGAGGCTGCC CCT - #CTCACCA           2088                                                                          Lys                                                                           #            2090                                                             - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 567 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pr - #o Leu His Ile Val Leu         #                 15                                                          - Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro Hi - #s Val Gln Lys Ser Val         #             30                                                              - Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gl - #y Ala Val Lys Phe Pro         #         45                                                                  - Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Se - #r Thr Cys Asp Asn Gln         #     60                                                                      - Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Se - #r Ile Cys Glu Lys Pro         # 80                                                                          - Gln Glu Val Cys Val Ala Val Trp Arg Lys As - #n Asp Glu Asn Ile Thr         #                 95                                                          - Leu Glu Thr Val Cys His Asp Pro Lys Leu Pr - #o Tyr His Asp Phe Ile         #           110                                                               - Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Me - #t Lys Glu Lys Lys Lys         #       125                                                                   - Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Se - #r Ser Asp Glu Cys Asn         #   140                                                                       - Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Th - #r Ser Asn Pro Asp Leu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Leu Leu Val Ile Phe Gln Val Thr Gly Ile Se - #r Leu Leu Pro Pro Leu         #               175                                                           - Gly Val Ala Ile Ser Val Ile Ile Ile Phe Ty - #r Cys Tyr Arg Val Asn         #           190                                                               - Arg Gln Gln Lys Leu Ser Ser Thr Trp Glu Th - #r Gly Lys Thr Arg Lys         #       205                                                                   - Leu Met Glu Phe Ser Glu His Cys Ala Ile Il - #e Leu Glu Asp Asp Arg         #   220                                                                       - Ser Asp Ile Ser Ser Thr Cys Ala Asn Asn Il - #e Asn His Asn Thr Glu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Leu Pro Ile Glu Leu Asp Thr Leu Val Gl - #y Lys Gly Arg Phe Ala         #               255                                                           - Glu Val Tyr Lys Ala Lys Leu Lys Gln Asn Th - #r Ser Glu Gln Phe Glu         #           270                                                               - Thr Val Ala Val Lys Ile Phe Pro Tyr Glu Gl - #u Tyr Ala Ser Trp Lys         #       285                                                                   - Thr Glu Lys Asp Ile Phe Ser Asp Ile Asn Le - #u Lys His Glu Asn Ile         #   300                                                                       - Leu Gln Phe Leu Thr Ala Glu Glu Arg Lys Th - #r Glu Leu Gly Lys Gln         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Tyr Trp Leu Ile Thr Ala Phe His Ala Lys Gl - #y Asn Leu Gln Glu Tyr         #               335                                                           - Leu Thr Arg His Val Ile Ser Trp Glu Asp Le - #u Arg Lys Leu Gly Ser         #           350                                                               - Ser Leu Ala Arg Gly Ile Ala His Leu His Se - #r Asp His Thr Pro Cys         #       365                                                                   - Gly Arg Pro Lys Met Pro Ile Val His Arg As - #p Leu Lys Ser Ser Asn         #   380                                                                       - Ile Leu Val Lys Asn Asp Leu Thr Cys Cys Le - #u Cys Asp Phe Gly Leu         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Ser Leu Arg Leu Asp Pro Thr Leu Ser Val As - #p Asp Leu Ala Asn Ser         #               415                                                           - Gly Gln Val Gly Thr Ala Arg Tyr Met Ala Pr - #o Glu Val Leu Glu Ser         #           430                                                               - Arg Met Asn Leu Glu Asn Ala Glu Ser Phe Ly - #s Gln Thr Asp Val Tyr         #       445                                                                   - Ser Met Ala Leu Val Leu Trp Glu Met Thr Se - #r Arg Cys Asn Ala Val         #   460                                                                       - Gly Glu Val Lys Asp Tyr Glu Pro Pro Phe Gl - #y Ser Lys Val Arg Glu         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - His Pro Cys Val Glu Ser Met Lys Asp Asn Va - #l Leu Arg Asp Arg Gly         #               495                                                           - Arg Pro Glu Ile Pro Ser Phe Trp Leu Asn Hi - #s Gln Gly Ile Gln Met         #           510                                                               - Val Cys Glu Thr Leu Thr Glu Cys Trp Asp Hi - #s Asp Pro Glu Ala Arg         #       525                                                                   - Leu Thr Ala Gln Cys Val Ala Glu Arg Phe Se - #r Glu Leu Glu His Leu         #   540                                                                       - Asp Arg Leu Ser Gly Arg Ser Cys Ser Glu Gl - #u Lys Ile Pro Glu Asp         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Gly Ser Leu Asn Thr Thr Lys                                                                 565                                                           __________________________________________________________________________

We claim:
 1. A method of altering TGF-β binding to TGF-β type IIreceptor on the surface of a cell, comprising combining with the cell apreparation consisting essentially of a soluble polypeptide comprisingthe amino acid sequence of the extracellular domain of a mammalian TGF-βtype II receptor protein, wherein the amino acid sequence of themammalian receptor protein is:(a) the amino acid sequence of the TGF-βtype II receptor protein of SEQ ID NO: 8, or (b) the amino acid sequenceof a TGF-β type II receptor protein encoded by mammalian DNA whichhybridizes to a probe having the sequence of the complement of SEQ IDNO: 7 under high stringency conditions; wherein the polypeptide and cellare combined under conditions appropriate for binding of the solubleTGF-β type II receptor to TGF-β.
 2. A method according to claim 1,wherein the mammalian TGF-β type II receptor protein has the amino acidsequence set forth in SEQ ID NO:
 8. 3. The method of claim 1, whereinTGF-β binding is inhibited.
 4. A method of altering TGF-β binding toTGF-β type II receptor on the surface of a cell, comprising combiningwith the cell a preparation consisting essentially of a solublepolypeptide comprising the amino acid sequence of the extracellulardomain of a mammalian TGF-β type II receptor protein, wherein themammalian receptor protein has the amino acid sequence encoded by:(a)the cDNA insert contained in the plasmid deposited under ATCC accessionnumber 209455, or (b) a cDNA molecule which is present in a mammalianlibrary and which hybridizes with a probe having the sequence of thecomplement of the coding sequence of the cDNA insert contained in theplasmid deposited under ATCC accession number 209455 under stringencyconditions sufficient to specifically identify the cDNA molecule in thelibrary;wherein the polypeptide and cell are combined under conditionsappropriate for binding of the soluble TGF-β type II receptor to TGF-β.5. A method according to claim 4, wherein the mammalian TGF-β type IIreceptor protein has the amino acid sequence encoded by the cDNA insertcontained in the plasmid deposited under ATCC accession number 209455.6. A method of altering TGF-β binding to TGF-β type II receptor on thesurface of a cell, comprising combining with the cell a preparationconsisting essentially of a polypeptide comprising part of the aminoacid sequence of a mammalian TGF-β type II receptor protein, wherein themammalian receptor protein has the amino acid sequence of:(a) the aminoacid sequence of the TGF-β type II receptor protein of SEQ ID NO: 8, or(b) the amino acid sequence encoded by mammalian DNA which hybridizes toa probe having the sequence of the complement of SEQ ID NO: 7 under highstringency conditions;wherein the amino acid sequence of the polypeptidecomprises a contiguous fragment of the mature mammalian type II receptorsequence which includes the TGF-β-binding site; wherein the polypeptidespecifically binds to TGF-β; and wherein the polypeptide and cell arecombined under conditions appropriate for binding of the soluble TGF-βtype II receptor to TGF-β.
 7. A method according to claim 6, wherein themammalian TGF-β type II receptor protein has the amino acid sequence setforth in SEQ ID NO:
 8. 8. A method according to claim 6, wherein thepolypeptide comprises the amino acid sequence of the extracellulardomain of the TGF-β type II receptor protein having the sequence of SEQID NO:
 8. 9. A method of altering TGF-β binding to TGF-β type IIreceptor on the surface of a cell, comprising combining with the cell apreparation consisting essentially of a polypeptide comprising part ofthe amino acid sequence of a mammalian TGF-β type II receptor protein,wherein the mammalian receptor protein has the amino acid sequenceencoded by:(a) the cDNA insert contained in the plasmid deposited underATCC accession number 209455, or (b) a cDNA molecule which is present ina mammalian library and which hybridizes with a probe having thesequence of the complement of the coding sequence of the cDNA insertcontained in the plasmid deposited under ATCC accession number 209455under stringency conditions sufficient to specifically identify the cDNAmolecule in the library;wherein the amino acid sequence of thepolypeptide comprises a contiguous fragment of the mature mammalian typeII receptor sequence which includes the TGF-β-binding site; wherein thepolypeptide specifically binds to TGF-β; and wherein the polypeptide andcell are combined under conditions appropriate for binding of thesoluble TGF-β type II receptor to TGF-β.
 10. A method according to claim9, wherein the mammalian TGF-β type II receptor protein has the aminoacid sequence encoded by the cDNA insert contained in the plasmiddeposited under ATCC accession number
 209455. 11. A method according toclaim 9, wherein the polypeptide comprises the amino acid sequence ofthe extracellular domain of the TGF-β type II receptor protein encodedby the cDNA insert contained in the plasmid deposited under ATCCaccession number
 209455. 12. A method of modulating the effects of TGF-βin a mammal, comprising administering to the mammal a polypeptidecomprising the amino acid sequence of a mammalian TGF-β type II receptorwherein the mammalian receptor has the amino acid sequence of:a) theamino acid sequence of the TGF-β type II receptor protein of SEQ ID NO:8, or b) the amino acid sequence encoded by mammalian DNA whichhybridizes under high stringency conditions to a probe having thesequence of the complement of SEQ ID NO: 7;wherein the polypeptide isadministered to the mammal in sufficient quantity to alter binding ofTGF-β to endogenous TGF-β type II receptors, type III receptors, orboth.
 13. A method according to claim 12, wherein the mammalian TGF-βtype II receptor protein has the amino acid sequence set forth in SEQ IDNO:
 8. 14. A method of modulating the effects of TGF-β in a mammal,comprising administering to the mammal a polypeptide comprising theamino acid sequence of a mammalian TGF-β type II receptor wherein themammalian receptor protein has the amino acid sequence encoded by:a) thecDNA insert contained in the plasmid deposited under ATCC accessionnumber 209455, or b) a cDNA molecule which is present in a mammalianlibrary and which hybridizes with a probe having the sequence of thecomplement of the coding sequence of the cDNA insert contained in theplasmid deposited under ATCC accession number 209455 under stringencyconditions sufficient to specifically identify the cDNA molecule in thelibrary;wherein the polypeptide is administered to the mammal insufficient quantity to alter binding of TGF-β to endogenous TGF-β typeII receptors, type III receptors, or both.
 15. A method according toclaim 14, wherein the mammalian TGF-β type II receptor protein has theamino acid sequence encoded by the cDNA insert contained in the plasmiddeposited under ATCC accession number
 209455. 16. A method of modulatingthe effects of TGF-β in a mammal, comprising administering to the mammala polypeptide comprising part of the amino acid sequence of a mammalianTGF-β type II receptor protein, wherein the mammalian receptor proteinhas the amino acid sequence of:a) the amino acid sequence of the TGF-βtype II receptor protein of SEQ ID NO: 8, or b) the amino acid sequenceencoded by mammalian DNA which hybridizes to a probe having the sequenceof the complement of SEQ ID NO: 7 under high stringencyconditions;wherein the amino acid sequence of the polypeptide comprisesa contiguous fragment of the mature mammalian type II receptor sequencewhich includes the TGF-β-binding site; wherein the polypeptidespecifically binds to TGF-β under conditions appropriate for binding ofthe TGF-β type II receptor to TGF-β; and wherein the polypeptide isadministered to the mammal in sufficient quantity to alter binding ofTGF-β to endogenous TGF-β type II receptors, type III receptors, orboth.
 17. A method according to claim 16, wherein the mammalian TGF-βtype II receptor protein has the amino acid sequence set forth in SEQ IDNO:
 8. 18. A method according to claim 16, wherein the polypeptidecomprises the amino acid sequence of the extracellular domain of theTGF-β type II receptor protein having the sequence of SEQ ID NO:
 8. 19.A method of modulating the effects of TGF-β in a mammal comprisingadministering to the mammal a polypeptide comprising part of the aminoacid sequence of a mammalian TGF-β type II receptor protein, wherein themammalian receptor protein has the amino acid sequence encoded by;a) thecDNA insert contained in the plasmid deposited under ATCC accessionnumber 209455, or b) a cDNA molecule which is present in a mammalianlibrary and which hybridizes with a probe having the sequence of thecomplement of the coding sequence of the cDNA insert contained in theplasmid deposited under ATCC accession number 209455 under stringencyconditions sufficient to specifically identify the cDNA molecule in thelibrary;wherein the amino acid sequence of the polypeptide comprises acontiguous fragment of the mature mammalian type II receptor sequencewhich includes the TGF-β-binding site; wherein the polypeptidespecifically binds to TGF-β under conditions appropriate for binding ofthe soluble TGF-β type II receptor to TGF-β; and wherein the polypeptideis administered to the mammal in sufficient quantity to alter binding ofTGF-β to endogenous TGF-β type II receptors, type III receptors, orboth.
 20. A method according to claim 19, wherein the mammalian TGF-βtype II receptor protein has the amino acid sequence encoded by the cDNAinsert contained in the plasmid deposited under ATCC accession number209455.
 21. A method according to claim 19, wherein the polypeptidecomprises the amino acid sequence of the extracellular domain of theTGF-β type II receptor protein encoded by the cDNA insert contained inthe plasmid deposited under ATCC accession number 209455.